G protein mediated signal transduction pathways are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. G protein mediated signal transduction occurs when an agonist binds selectively to its heptahelical receptor leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha (Ga), beta (Gb) and gamma (Gg) subunits, and when activated they are able to regulate the activity of specific effectors. Most cells harbor multiple G protein-mediated signaling pathways with the potential to work at cross purposes unless they are appropriately segregated from one another. Mounting evidence suggests that this is achieved by assembling receptors, G proteins and effectors into signaling complexes. Two fluorescence based techniques are being used to investigate when and where these complexes are formed in living cells. These techniques, known as bioluminescent resonance energy transfer (BRET), and bimolecular fluorescence complementation (BiFC), can provide both spatial and temporal information about the formation and dissolution of protein complexes. BRET involves the exogenous expression of fusion proteins tagged with either luciferase (Luc) or a fluorescent moiety. The fluorescent moiety can be a fluorescent protein, such as GFP or YFP, or the peptide motif CCPGCC that is capable of binding biarsenical derivatives of fluorescent compound (ie. FlAsH). BRET occurs when the bioluminescent energy of the Luc tag is transferred to the fluorescent tag causing it to fluoresce. This only occurs if the tags are juxtaposed (less than 100 angstroms apart) because the fusion proteins associate to form a complex. BiFC is based on the fact that complementary N- and C-terminal fragments of YFP (YN and YC, respectively) are not themselves fluorescent, but will reconstitute a fluorescent YFP molecule if they are brought together by being fused to proteins that associate to form a complex.[unreadable] [unreadable] The heptahelical beta2 adrenergic receptor (b2AR) and the dopamine D4.2 receptor (D4.2R) trigger the activation of G proteins leading to the regulation of effectors including adenylyl cyclase (AC) and G protein coupled inwardly rectifying K+ (Kir3) channels. When these proteins were tagged for BRET and BiFC experiments they retained their biological activity. BRET was used to show that the D4.2R forms as complex with AC. Because the D4.2R is inactivated by fusion with either Luc or a fluorescent protein these experiments require that the CCPGCC motif be incorporated into the receptor. FlAsH binding this motif makes the D4.2R an acceptor for resonance energy transfer from AC-Luc in BRET experiments. BRET experiments also showed that the b2AR forms a complex with AC and with the Kir3 channel subunit, Kir3.1. These complexes exist in the absence of signal transduction and persist during signal transduction. BRET as well as co-immunoprecipitation experiments were also used to show that G protein subunits form complexes with the b2AR, AC and Kir3.1. BRET between the G protein subunits and these signaling proteins was affected by a receptor agonist. Experiments designed to probe the nature of the agonist-induced effects indicated that they were caused by altered conformations within a protein complex that remains intact.[unreadable] [unreadable] Gb and Gg form a stable heterodimer (Gbg). BiFC occurs when Gb tagged with YN and Gg tagged with YC heterodimerize to bring YN and YC together so that a fluorescent YPF is reconstituted. When this BiFC pair is co-expressed with either the b2AR-Luc or a Luc-tagged effector (AC-Luc or Kir3.1-Luc) BRET occurred indicating the simultaneous presence of three different proteins in the same signaling complex. The technique of combining BiFC and BRET is now being used to show that b2AR, G protein subunits and effectors are all simultaneously part of the same complex in living cells.[unreadable] [unreadable] Experimental evidence also supports the hypothesis that G protein-mediated signaling complexes are formed before they reach the plasma membrane. BRET together with subcellular fractionation demonstrated that a complex of AC and the b2AR are present on intracellular membranes. Further, dominant-negative (DN) Rab1 and Sar1 GTPases which block anterograde trafficking out of the endoplasmic reticulum (ER) have no effect on either AC/b2AR or AC/Gbg protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Ga subunits in AC signaling complexes suggesting Ga becomes part of the complex at some point beyond the ER. In summary our data support the hypothesis that the heptahelical receptors, G proteins and effectors are assembled into complexes before being transported to the plasma membrane, and that these complexes persist when the signal transduction pathway is activated by an agonist. This arrangement helps to explain the specificity and efficacy that is often observed during G protein mediated signal transduction.